Project 2 Project Summary Abstract The implication of extracellular membrane vesicles such as exosomes, and membrane protrusions such as tunneling nanotubes, in intercellular trafficking networks opens a new perspective in understanding tumor development and progression. The ubiquitin system is one of the central regulators of biogenesis and function of exosomes and nanotubes, and infection with tumor viruses including Epstein-Barr Virus (EBV) results in deregulation of cellular functions by manipulation of this system during cell transformation. Studies in recent years clearly demonstrate that the spectrum of potential functions of the small evolutionarily conserved family of Ubiquitin C-terminal Hydrolases (UCHs) is much wider than was suspected. Among them, UCH-l1 is of special interest: recent studies including ours demonstrate that this unique deubiquitinase is closely involved not only in cell transformation and in primary tumor formation, but is a main regulator of cancer progression as well. Based directly on our previous studies and substantial preliminary data, we hypothesize that UCH-L1 is a major regulator of ubiquitin-dependent processes of intra- and inter-cellular trafficking in EBV-positive cancers. In Aim I, we will analyze how distinct biochemical functions of UCH-L1 are required for exosome biogenesis and sorting, and how the EBV major oncogene, Latent Membrane Protein 1 (LMP1), is involved in these processes. In Aim II, based on our recent discovery that N-cadherin is highly expressed in EBV-driven B-cell lymphomas, and co-localizes with UCH-L1 in these cells, we will explore the role of N-cadherin-based complexes in intercellular trafficking of pro-metastatic factors produced by EBV-positive cancer cells. The results in this Aim will clarify how tunneling nanotubes and exosomes produced by EBV-transformed cells change the tumor microenvironment by transferring pro-invasive factors to tumor stromal tissues. In Aim III we will investigate whether inhibition of UCH-L1 activity with specific small-molecule inhibitors that have anti- tumorigenic effects in cell culture will also be active in vivo against EBV B-cell lymphogenesis in humanized mice. We will determine whether UCH-L1 DUB activity is required for EBV-induced immortalization of normal human B-cells, and whether inhibition of UCH-L1 affects expression and function of EBV genes in latently infected cells as well as during viral reactivation. EBV is tightly linked to several highly invasive malignancies of lymphoid and epithelial origin; treatment of patients with these malignancies poses unique challenges, and outcomes remain poor. Since recent study has demonstrated a profound anti-metastatic effect of such UCH-L1 inhibitor in a mouse model of invasive carcinoma, specific inhibitors of UCH-L1 enzymatic activity may offer an adjunct to existing therapies.